JP2022537477A - 機能的エレメントの同定方法 - Google Patents
機能的エレメントの同定方法 Download PDFInfo
- Publication number
- JP2022537477A JP2022537477A JP2021557446A JP2021557446A JP2022537477A JP 2022537477 A JP2022537477 A JP 2022537477A JP 2021557446 A JP2021557446 A JP 2021557446A JP 2021557446 A JP2021557446 A JP 2021557446A JP 2022537477 A JP2022537477 A JP 2022537477A
- Authority
- JP
- Japan
- Prior art keywords
- amino acid
- score
- deletion
- deletions
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 185
- 150000001413 amino acids Chemical class 0.000 claims description 549
- 230000035772 mutation Effects 0.000 claims description 380
- 230000037430 deletion Effects 0.000 claims description 373
- 238000012217 deletion Methods 0.000 claims description 373
- 108090000623 proteins and genes Proteins 0.000 claims description 311
- 108020005004 Guide RNA Proteins 0.000 claims description 203
- 230000008859 change Effects 0.000 claims description 187
- 102000004169 proteins and genes Human genes 0.000 claims description 166
- 238000012216 screening Methods 0.000 claims description 136
- 230000008685 targeting Effects 0.000 claims description 88
- 239000013598 vector Substances 0.000 claims description 67
- 108020004414 DNA Proteins 0.000 claims description 66
- 238000009826 distribution Methods 0.000 claims description 58
- 239000002299 complementary DNA Substances 0.000 claims description 57
- 229940079593 drug Drugs 0.000 claims description 51
- 239000003814 drug Substances 0.000 claims description 51
- 230000001413 cellular effect Effects 0.000 claims description 49
- 239000003053 toxin Substances 0.000 claims description 49
- 231100000765 toxin Toxicity 0.000 claims description 49
- 238000003776 cleavage reaction Methods 0.000 claims description 48
- 230000007017 scission Effects 0.000 claims description 48
- 102100035102 E3 ubiquitin-protein ligase MYCBP2 Human genes 0.000 claims description 47
- 238000012163 sequencing technique Methods 0.000 claims description 44
- 239000012634 fragment Substances 0.000 claims description 43
- 230000014509 gene expression Effects 0.000 claims description 43
- 230000006870 function Effects 0.000 claims description 35
- 239000013612 plasmid Substances 0.000 claims description 33
- 238000011144 upstream manufacturing Methods 0.000 claims description 27
- 238000002703 mutagenesis Methods 0.000 claims description 25
- 231100000350 mutagenesis Toxicity 0.000 claims description 25
- 239000002773 nucleotide Substances 0.000 claims description 24
- 125000003729 nucleotide group Chemical group 0.000 claims description 24
- 230000008569 process Effects 0.000 claims description 24
- 230000001105 regulatory effect Effects 0.000 claims description 24
- 238000004364 calculation method Methods 0.000 claims description 18
- 238000013518 transcription Methods 0.000 claims description 16
- 230000035897 transcription Effects 0.000 claims description 16
- 230000001939 inductive effect Effects 0.000 claims description 13
- 230000003612 virological effect Effects 0.000 claims description 13
- 238000013507 mapping Methods 0.000 claims description 12
- 102000040430 polynucleotide Human genes 0.000 claims description 12
- 108091033319 polynucleotide Proteins 0.000 claims description 12
- 239000002157 polynucleotide Substances 0.000 claims description 12
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 11
- 230000001965 increasing effect Effects 0.000 claims description 10
- 239000003623 enhancer Substances 0.000 claims description 7
- 238000001914 filtration Methods 0.000 claims description 7
- 108091027963 non-coding RNA Proteins 0.000 claims description 6
- 102000042567 non-coding RNA Human genes 0.000 claims description 6
- 239000013600 plasmid vector Substances 0.000 claims description 5
- 230000007423 decrease Effects 0.000 claims description 3
- 230000032537 response to toxin Effects 0.000 claims description 3
- 230000003247 decreasing effect Effects 0.000 claims 4
- 230000004075 alteration Effects 0.000 claims 2
- 230000011559 double-strand break repair via nonhomologous end joining Effects 0.000 claims 1
- 235000001014 amino acid Nutrition 0.000 description 537
- 210000004027 cell Anatomy 0.000 description 280
- 108091027544 Subgenomic mRNA Proteins 0.000 description 250
- 235000018102 proteins Nutrition 0.000 description 131
- 108091033409 CRISPR Proteins 0.000 description 128
- 101000735881 Homo sapiens Proteasome subunit beta type-5 Proteins 0.000 description 116
- 102100036127 Proteasome subunit beta type-5 Human genes 0.000 description 116
- 102100031463 Serine/threonine-protein kinase PLK1 Human genes 0.000 description 84
- 108010056274 polo-like kinase 1 Proteins 0.000 description 84
- 238000010354 CRISPR gene editing Methods 0.000 description 81
- 230000000694 effects Effects 0.000 description 71
- 101800001649 Heparin-binding EGF-like growth factor Proteins 0.000 description 65
- 108010053187 Diphtheria Toxin Proteins 0.000 description 64
- 102000016607 Diphtheria Toxin Human genes 0.000 description 64
- 102400001369 Heparin-binding EGF-like growth factor Human genes 0.000 description 57
- 229960001467 bortezomib Drugs 0.000 description 57
- GXJABQQUPOEUTA-RDJZCZTQSA-N bortezomib Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)B(O)O)NC(=O)C=1N=CC=NC=1)C1=CC=CC=C1 GXJABQQUPOEUTA-RDJZCZTQSA-N 0.000 description 57
- 108700012359 toxins Proteins 0.000 description 46
- 102100031323 Anthrax toxin receptor 1 Human genes 0.000 description 45
- 238000011156 evaluation Methods 0.000 description 44
- 101000796095 Homo sapiens Anthrax toxin receptor 1 Proteins 0.000 description 43
- 108020004705 Codon Proteins 0.000 description 42
- XQVVPGYIWAGRNI-JOCHJYFZSA-N bi-2536 Chemical compound N1([C@@H](C(N(C)C2=CN=C(NC=3C(=CC(=CC=3)C(=O)NC3CCN(C)CC3)OC)N=C21)=O)CC)C1CCCC1 XQVVPGYIWAGRNI-JOCHJYFZSA-N 0.000 description 42
- 238000006467 substitution reaction Methods 0.000 description 42
- 230000027455 binding Effects 0.000 description 41
- 102100028757 Chondroitin sulfate proteoglycan 4 Human genes 0.000 description 40
- 101000916489 Homo sapiens Chondroitin sulfate proteoglycan 4 Proteins 0.000 description 40
- 238000007481 next generation sequencing Methods 0.000 description 40
- 238000009739 binding Methods 0.000 description 39
- 238000012408 PCR amplification Methods 0.000 description 36
- 238000011282 treatment Methods 0.000 description 35
- 230000035945 sensitivity Effects 0.000 description 34
- 230000004853 protein function Effects 0.000 description 32
- 125000003275 alpha amino acid group Chemical group 0.000 description 29
- 150000001875 compounds Chemical class 0.000 description 27
- 238000004458 analytical method Methods 0.000 description 24
- WYWHKKSPHMUBEB-UHFFFAOYSA-N tioguanine Chemical compound N1C(N)=NC(=S)C2=C1N=CN2 WYWHKKSPHMUBEB-UHFFFAOYSA-N 0.000 description 24
- 230000001988 toxicity Effects 0.000 description 24
- 231100000419 toxicity Toxicity 0.000 description 24
- 108020003175 receptors Proteins 0.000 description 22
- 102000005962 receptors Human genes 0.000 description 22
- 108020004485 Nonsense Codon Proteins 0.000 description 20
- 230000037431 insertion Effects 0.000 description 20
- 238000003780 insertion Methods 0.000 description 20
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 18
- 206010059866 Drug resistance Diseases 0.000 description 18
- 239000002246 antineoplastic agent Substances 0.000 description 18
- 108091026890 Coding region Proteins 0.000 description 17
- 239000000047 product Substances 0.000 description 17
- 230000002378 acidificating effect Effects 0.000 description 16
- 230000037434 nonsense mutation Effects 0.000 description 16
- 238000005259 measurement Methods 0.000 description 15
- 101100464293 Caenorhabditis elegans plk-1 gene Proteins 0.000 description 14
- 102000012545 EGF-like domains Human genes 0.000 description 14
- 108050002150 EGF-like domains Proteins 0.000 description 14
- 238000003556 assay Methods 0.000 description 14
- 230000037433 frameshift Effects 0.000 description 14
- 238000004422 calculation algorithm Methods 0.000 description 13
- 108700028369 Alleles Proteins 0.000 description 12
- 101150063416 add gene Proteins 0.000 description 12
- 230000003321 amplification Effects 0.000 description 12
- 230000003013 cytotoxicity Effects 0.000 description 12
- 231100000135 cytotoxicity Toxicity 0.000 description 12
- 238000003199 nucleic acid amplification method Methods 0.000 description 12
- 230000014616 translation Effects 0.000 description 11
- 241000894006 Bacteria Species 0.000 description 10
- 241000713666 Lentivirus Species 0.000 description 10
- 230000030833 cell death Effects 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 10
- 230000002068 genetic effect Effects 0.000 description 10
- 230000012010 growth Effects 0.000 description 10
- 102000039446 nucleic acids Human genes 0.000 description 10
- 108020004707 nucleic acids Proteins 0.000 description 10
- 150000007523 nucleic acids Chemical class 0.000 description 10
- 238000010361 transduction Methods 0.000 description 10
- 230000026683 transduction Effects 0.000 description 10
- 241000219315 Spinacia Species 0.000 description 9
- 235000009337 Spinacia oleracea Nutrition 0.000 description 9
- 229940041181 antineoplastic drug Drugs 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 241001270131 Agaricus moelleri Species 0.000 description 8
- 231100000699 Bacterial toxin Toxicity 0.000 description 8
- 108700039887 Essential Genes Proteins 0.000 description 8
- 239000000688 bacterial toxin Substances 0.000 description 8
- 238000007405 data analysis Methods 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 238000013519 translation Methods 0.000 description 8
- 230000034994 death Effects 0.000 description 7
- 210000003527 eukaryotic cell Anatomy 0.000 description 7
- 230000006780 non-homologous end joining Effects 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 238000010200 validation analysis Methods 0.000 description 7
- 108020004635 Complementary DNA Proteins 0.000 description 6
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 6
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 6
- 241000124008 Mammalia Species 0.000 description 6
- 108091034117 Oligonucleotide Proteins 0.000 description 6
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 238000004520 electroporation Methods 0.000 description 6
- 231100000221 frame shift mutation induction Toxicity 0.000 description 6
- 238000010448 genetic screening Methods 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 6
- 230000003993 interaction Effects 0.000 description 6
- 108020004999 messenger RNA Proteins 0.000 description 6
- 238000004806 packaging method and process Methods 0.000 description 6
- 108020001580 protein domains Proteins 0.000 description 6
- 230000000717 retained effect Effects 0.000 description 6
- 238000010839 reverse transcription Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 230000004083 survival effect Effects 0.000 description 6
- 238000012546 transfer Methods 0.000 description 6
- 206010063409 Acarodermatitis Diseases 0.000 description 5
- 102000018251 Hypoxanthine Phosphoribosyltransferase Human genes 0.000 description 5
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 description 5
- 241000447727 Scabies Species 0.000 description 5
- 238000013459 approach Methods 0.000 description 5
- 238000004140 cleaning Methods 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 208000005687 scabies Diseases 0.000 description 5
- 230000009870 specific binding Effects 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- 241000193738 Bacillus anthracis Species 0.000 description 4
- 238000010453 CRISPR/Cas method Methods 0.000 description 4
- 108700022831 Clostridium difficile toxB Proteins 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 4
- 241000206602 Eukaryota Species 0.000 description 4
- 241000233866 Fungi Species 0.000 description 4
- 102220639700 Leptin_R78L_mutation Human genes 0.000 description 4
- 101710163270 Nuclease Proteins 0.000 description 4
- 108091028113 Trans-activating crRNA Proteins 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 239000003242 anti bacterial agent Substances 0.000 description 4
- 229940088710 antibiotic agent Drugs 0.000 description 4
- 230000003115 biocidal effect Effects 0.000 description 4
- 210000004899 c-terminal region Anatomy 0.000 description 4
- 239000003560 cancer drug Substances 0.000 description 4
- 238000000749 co-immunoprecipitation Methods 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 239000004121 copper complexes of chlorophylls and chlorophyllins Substances 0.000 description 4
- 230000001086 cytosolic effect Effects 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 230000005782 double-strand break Effects 0.000 description 4
- 238000007877 drug screening Methods 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- 239000012091 fetal bovine serum Substances 0.000 description 4
- 108020001507 fusion proteins Proteins 0.000 description 4
- 102000037865 fusion proteins Human genes 0.000 description 4
- 238000010362 genome editing Methods 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 239000002502 liposome Substances 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 238000010369 molecular cloning Methods 0.000 description 4
- 230000009437 off-target effect Effects 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 102220057745 rs730881370 Human genes 0.000 description 4
- 102220082272 rs863223860 Human genes 0.000 description 4
- 239000000344 soap Substances 0.000 description 4
- 230000002463 transducing effect Effects 0.000 description 4
- 238000001712 DNA sequencing Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000003596 drug target Substances 0.000 description 3
- 230000004077 genetic alteration Effects 0.000 description 3
- 231100000118 genetic alteration Toxicity 0.000 description 3
- 238000003306 harvesting Methods 0.000 description 3
- 210000005260 human cell Anatomy 0.000 description 3
- 238000009396 hybridization Methods 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 125000006850 spacer group Chemical group 0.000 description 3
- NBWRJAOOMGASJP-UHFFFAOYSA-N 2-(3,5-diphenyl-1h-tetrazol-1-ium-2-yl)-4,5-dimethyl-1,3-thiazole;bromide Chemical compound [Br-].S1C(C)=C(C)N=C1N1N(C=2C=CC=CC=2)N=C(C=2C=CC=CC=2)[NH2+]1 NBWRJAOOMGASJP-UHFFFAOYSA-N 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- 241000238421 Arthropoda Species 0.000 description 2
- 238000012935 Averaging Methods 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 2
- 241000195493 Cryptophyta Species 0.000 description 2
- 241000450599 DNA viruses Species 0.000 description 2
- 206010064571 Gene mutation Diseases 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101000615488 Homo sapiens Methyl-CpG-binding domain protein 2 Proteins 0.000 description 2
- 101000871708 Homo sapiens Proheparin-binding EGF-like growth factor Proteins 0.000 description 2
- 239000006142 Luria-Bertani Agar Substances 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 102100021299 Methyl-CpG-binding domain protein 2 Human genes 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 108010021466 Mutant Proteins Proteins 0.000 description 2
- 102000008300 Mutant Proteins Human genes 0.000 description 2
- 241000244206 Nematoda Species 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 102100035593 POU domain, class 2, transcription factor 1 Human genes 0.000 description 2
- 101710084414 POU domain, class 2, transcription factor 1 Proteins 0.000 description 2
- 241000288906 Primates Species 0.000 description 2
- 102220587727 Protein FAM102A_M104A_mutation Human genes 0.000 description 2
- 238000002123 RNA extraction Methods 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 108700008625 Reporter Genes Proteins 0.000 description 2
- 241000283984 Rodentia Species 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 241000283907 Tragelaphus oryx Species 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 150000001295 alanines Chemical class 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000004166 bioassay Methods 0.000 description 2
- 238000011325 biochemical measurement Methods 0.000 description 2
- 238000003766 bioinformatics method Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 229910021538 borax Inorganic materials 0.000 description 2
- 239000001678 brown HT Substances 0.000 description 2
- 101150102092 ccdB gene Proteins 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000003915 cell function Effects 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 229940044683 chemotherapy drug Drugs 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 230000009918 complex formation Effects 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000012937 correction Methods 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 210000001840 diploid cell Anatomy 0.000 description 2
- 229960003722 doxycycline Drugs 0.000 description 2
- XQTWDDCIUJNLTR-CVHRZJFOSA-N doxycycline monohydrate Chemical compound O.O=C1C2=C(O)C=CC=C2[C@H](C)[C@@H]2C1=C(O)[C@]1(O)C(=O)C(C(N)=O)=C(O)[C@@H](N(C)C)[C@@H]1[C@H]2O XQTWDDCIUJNLTR-CVHRZJFOSA-N 0.000 description 2
- 241001493065 dsRNA viruses Species 0.000 description 2
- 230000012202 endocytosis Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 239000003797 essential amino acid Substances 0.000 description 2
- 235000020776 essential amino acid Nutrition 0.000 description 2
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 210000002816 gill Anatomy 0.000 description 2
- 239000008241 heterogeneous mixture Substances 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 102000043417 human HBEGF Human genes 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 238000000126 in silico method Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 238000005304 joining Methods 0.000 description 2
- 230000001665 lethal effect Effects 0.000 description 2
- 238000002898 library design Methods 0.000 description 2
- 230000004777 loss-of-function mutation Effects 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000000869 mutational effect Effects 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 230000009120 phenotypic response Effects 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 238000007781 pre-processing Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000001243 protein synthesis Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000002708 random mutagenesis Methods 0.000 description 2
- 230000008263 repair mechanism Effects 0.000 description 2
- 102200115280 rs121918070 Human genes 0.000 description 2
- 102220151559 rs886060514 Human genes 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 239000004328 sodium tetraborate Substances 0.000 description 2
- 235000010339 sodium tetraborate Nutrition 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 230000009897 systematic effect Effects 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 229960003087 tioguanine Drugs 0.000 description 2
- 230000024033 toxin binding Effects 0.000 description 2
- 239000013603 viral vector Substances 0.000 description 2
- 101150096316 5 gene Proteins 0.000 description 1
- -1 HPRT1 is 1.75×10 5 Proteins 0.000 description 1
- 238000012220 PCR site-directed mutagenesis Methods 0.000 description 1
- 101150048541 PSMB5 gene Proteins 0.000 description 1
- 230000007022 RNA scission Effects 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- MYHSVHWQEVDFQT-JOOHCUNXSA-N [(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] (1e)-3-hydroxy-n-sulfooxypent-4-enimidothioate Chemical compound OC[C@H]1O[C@@H](S\C(CC(O)C=C)=N\OS(O)(=O)=O)[C@H](O)[C@@H](O)[C@@H]1O MYHSVHWQEVDFQT-JOOHCUNXSA-N 0.000 description 1
- 238000003570 cell viability assay Methods 0.000 description 1
- 238000010668 complexation reaction Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000009650 gentamicin protection assay Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1079—Screening libraries by altering the phenotype or phenotypic trait of the host
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/04—Libraries containing only organic compounds
- C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B35/00—ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
- G16B35/10—Design of libraries
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B35/00—ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
- G16B35/20—Screening of libraries
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/111—General methods applicable to biologically active non-coding nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2320/00—Applications; Uses
- C12N2320/10—Applications; Uses in screening processes
- C12N2320/11—Applications; Uses in screening processes for the determination of target sites, i.e. of active nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2330/00—Production
- C12N2330/30—Production chemically synthesised
- C12N2330/31—Libraries, arrays
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Computational Biology (AREA)
- Plant Pathology (AREA)
- Library & Information Science (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Evolutionary Biology (AREA)
- Medical Informatics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Theoretical Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2019079729 | 2019-03-26 | ||
CNPCT/CN2019/079729 | 2019-03-26 | ||
PCT/CN2020/081283 WO2020192712A1 (en) | 2019-03-26 | 2020-03-26 | Method for identifying functional elements |
Publications (1)
Publication Number | Publication Date |
---|---|
JP2022537477A true JP2022537477A (ja) | 2022-08-26 |
Family
ID=72611084
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2021557446A Pending JP2022537477A (ja) | 2019-03-26 | 2020-03-26 | 機能的エレメントの同定方法 |
Country Status (8)
Country | Link |
---|---|
US (1) | US20220186210A1 (zh) |
EP (1) | EP3947788A4 (zh) |
JP (1) | JP2022537477A (zh) |
KR (1) | KR20220004980A (zh) |
CN (1) | CN113939617A (zh) |
AU (1) | AU2020248911B2 (zh) |
CA (1) | CA3134400A1 (zh) |
WO (1) | WO2020192712A1 (zh) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3663310A4 (en) | 2017-08-04 | 2021-08-11 | Peking University | TALE-RVD WITH SPECIFIC DETECTION OF A DNA BASE MODIFIED BY METHYLATION AND APPLICATION OF IT |
US11624077B2 (en) | 2017-08-08 | 2023-04-11 | Peking University | Gene knockout method |
CR20220063A (es) | 2019-07-12 | 2022-07-22 | Univ Beijing | Edición de ácido ribonucleico (arn) dirigido aprovechando la adenosina desaminasa que actúa sobre ácido ribonucleico endógeno (adar) utilizando ácidos ribonucleicos (arn) modificados genéticamente |
KR20240003760A (ko) * | 2022-06-29 | 2024-01-09 | 서울대학교산학협력단 | RNA 안정성 또는 mRNA 번역 증가용 신규 조절 엘리먼트,이와 상호작용하는 ZCCHC2, 및 이들의 용도 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016182893A1 (en) * | 2015-05-08 | 2016-11-17 | Teh Broad Institute Inc. | Functional genomics using crispr-cas systems for saturating mutagenesis of non-coding elements, compositions, methods, libraries and applications thereof |
WO2017219027A1 (en) * | 2016-06-17 | 2017-12-21 | The Broad Institute Inc. | Type vi crispr orthologs and systems |
JP2018524018A (ja) * | 2015-05-08 | 2018-08-30 | ザ チルドレンズ メディカル センター コーポレイション | 胎児型ヘモグロビン再誘導のための、bcl11aエンハンサー機能性領域を標的とする方法 |
WO2018191388A1 (en) * | 2017-04-12 | 2018-10-18 | The Broad Institute, Inc. | Novel type vi crispr orthologs and systems |
-
2020
- 2020-03-26 EP EP20777188.2A patent/EP3947788A4/en not_active Withdrawn
- 2020-03-26 WO PCT/CN2020/081283 patent/WO2020192712A1/en unknown
- 2020-03-26 AU AU2020248911A patent/AU2020248911B2/en active Active
- 2020-03-26 KR KR1020217034687A patent/KR20220004980A/ko not_active Application Discontinuation
- 2020-03-26 JP JP2021557446A patent/JP2022537477A/ja active Pending
- 2020-03-26 CA CA3134400A patent/CA3134400A1/en active Pending
- 2020-03-26 US US17/593,811 patent/US20220186210A1/en active Pending
- 2020-03-26 CN CN202080024624.0A patent/CN113939617A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016182893A1 (en) * | 2015-05-08 | 2016-11-17 | Teh Broad Institute Inc. | Functional genomics using crispr-cas systems for saturating mutagenesis of non-coding elements, compositions, methods, libraries and applications thereof |
JP2018524018A (ja) * | 2015-05-08 | 2018-08-30 | ザ チルドレンズ メディカル センター コーポレイション | 胎児型ヘモグロビン再誘導のための、bcl11aエンハンサー機能性領域を標的とする方法 |
WO2017219027A1 (en) * | 2016-06-17 | 2017-12-21 | The Broad Institute Inc. | Type vi crispr orthologs and systems |
WO2018191388A1 (en) * | 2017-04-12 | 2018-10-18 | The Broad Institute, Inc. | Novel type vi crispr orthologs and systems |
Non-Patent Citations (1)
Title |
---|
細胞, vol. 51, no. 3, JPN6023022007, 20 March 2019 (2019-03-20), pages 21 - 24, ISSN: 0005077616 * |
Also Published As
Publication number | Publication date |
---|---|
AU2020248911B2 (en) | 2022-12-15 |
US20220186210A1 (en) | 2022-06-16 |
EP3947788A1 (en) | 2022-02-09 |
AU2020248911A1 (en) | 2021-11-04 |
CN113939617A (zh) | 2022-01-14 |
EP3947788A4 (en) | 2022-06-08 |
WO2020192712A1 (en) | 2020-10-01 |
KR20220004980A (ko) | 2022-01-12 |
CA3134400A1 (en) | 2020-10-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11098326B2 (en) | Using RNA-guided FokI nucleases (RFNs) to increase specificity for RNA-guided genome editing | |
JP2022537477A (ja) | 機能的エレメントの同定方法 | |
KR102598819B1 (ko) | 서열결정에 의해 평가된 DSB의 게놈 전체에 걸친 비편향된 확인 (GUIDE-Seq) | |
US10011850B2 (en) | Using RNA-guided FokI Nucleases (RFNs) to increase specificity for RNA-Guided Genome Editing | |
CN113646434B (zh) | 使用加标签的向导rna构建体进行高效基因筛选的组合物和方法 | |
JP2018532419A (ja) | CRISPR−Cas sgRNAライブラリー | |
CN110520528A (zh) | 高保真性cas9变体及其应用 | |
JP7308380B2 (ja) | 遺伝子編集技術を用いたインビトロ部位特異的変異導入のための方法 | |
EP3519570B1 (en) | Method to analyze and optimize gene editing modules and delivery approaches | |
JP2019514379A (ja) | Rna誘導型ヌクレアーゼ活性のインビボ高スループット評価のための方法 | |
Moyer et al. | Generation of a conditional analog-sensitive kinase in human cells using CRISPR/Cas9-mediated genome engineering | |
Malina et al. | Adapting CRISPR/Cas9 for functional genomics screens | |
Karagyaur et al. | Practical recommendations for improving efficiency and accuracy of the CRISPR/Cas9 genome editing system | |
Escudero et al. | Primary and promiscuous functions coexist during evolutionary innovation through whole protein domain acquisitions | |
CN114729011A (zh) | 新型crispr dna靶向酶及系统 | |
US20190218544A1 (en) | Gene editing, identifying edited cells, and kits for use therein | |
Cebrailoglu et al. | CRISPR-Cas: removing boundaries of the nature | |
CN111748848B (zh) | 鉴定功能元件的方法 | |
WO2024119052A2 (en) | Genomic cryptography | |
Escudero García-Calderón et al. | Primary and promiscuous functions coexist during evolutionary innovation through whole protein domain acquisitions | |
US20060123491A1 (en) | Method for a (high through-put) screening detection of genetic modifications in genome engineering |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20220509 |
|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20220509 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20230606 |
|
A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20240109 |